One of the central issues in biology is the question of how telomeres are involved in genetic stability. Recombinational telomere elongation (RTE) is a mechanism by which cells elongate their telomeres in the absence of telomerase activity. Human ALT (alternative lengthening of telomeres) tumors depend upon RTE to lengthen telomeres and continue cellular division when telomerase is absent, as is the case in most human somatic cells. One of the components involved in telomere maintenance and homeostasis is Mre11, a multifunctional protein whose primary role is the recognition and repair of double-strand breaks. The long-term goal of the proposed research is to determine the mechanism by which cells undergo recombinational telomere elongation (RTE) and to apply this knowledge to the treatment of human cancers, such as osteosarcomas, caused by ALT cells. The specific objective of this project is to more clearly characterize the specific recombinational mechanism responsible for telomere elongation in yeast. These experiments use the novel gain-of-function allele, mre11- A470T, which is capable of inducing an unusual variation of telomeric recombination in telomerase-negative cells. This variation is responsible for an observed bypass of senescence in telomerase-negative, mre11-A470T cells. The central hypothesis is that RTE in yeast is accomplished via the break-induced replication (BIR) pathway and that the mre11-A470T allele induces aberrant telomeric BIR events. This alteration of the BIR pathway may occur by a generalized or specific enhancement of the activity, regulation, or specificity of BIR enzymatic machinery. The rationale behind the proposed experiments is that study of mre11-A470T provides an excellent and unique opportunity to probe the regulation of telomere recombination by Mre11. The significance of this work is that it will provide us with a greater understanding of the regulation, requirements, and characteristics of recombinational telomere elongation and the role of Mre11 in this process. This information will be essential to our understanding and treatment of the cancer progression of human ALT cells. PUBLIC HEALTH RELEVANCE: In human somatic cells, telomerase, the enzyme responsible for de novo telomere addition, is expressed in limited amounts, resulting in the gradual shortening of telomeres and replicative senescence. The lengthening of telomeres through telomerase-independent, recombinational mechanisms occur in approximately 10-15% of human cancers. Understanding the mechanism of this process is essential in the fight against cancerous human ALT (alternative lengthening of telomeres) cells. The proposed research is relevant to public health in that it will increase our understanding of the mechanisms of recombinational telomere elongation, particularly with regard to the role of Mre11 in the regulation of break-induced replication and telomere elongation. This information will be applicable to the further understanding of the mechanisms of survival of human ALT cells. This project is relevant to the mission of the NIH in that it seeks to increase our fundamental knowledge regarding the regulation of telomere elongation through the MRN complex and its connection to cellular senescence and survival.